Like other organs, the skin is well organized into histologically defined tissues to facilitate the performance of its various functions. The skin is divided into two main layers, the surface epithelium, epidermis, and the underlying connective tissue layer, dermis (Topical Drug Delivery Formulations, Eds. D. W. Osborne, A. H. Amann, Marcel Dekker, Inc. pages 87-91).
The first layer, epidermis is a keratinizing epithelium composed of several distinct cell populations. The keratinocytes represent more than 90% of the cells within the epidermis. These cells differentiate into the nonviable, flattened corneocytes of the stratum corneum, the outer most layer of the epidermis. Their interlocking plate-like structure provides the major barrier function for the skin.
Epidermal differentiation includes four distinct cellular events, one of them is the generation of neutral lipid-enriched intercellular domains, resulting from the secretion of distinctive structures named lamellar bodies. A lamellar body is a secretory organelle that is considered the central actor in the formation of the intercellular compartment that constitutes from 10% to 40% of the total volume of this tissue. Lamellar body exocytosis may contribute a large reservoir of surface area that could explain the stratum corneum's remarkable water holding capacity. Lamellar bodies appear to contain three types of materials: sugars, in the form of glycosphingolipids and glycoproteins; free sterols and phospholipids; and hydrolytic enzymes (acid phosphatase, proteases, lipases and glycosidases) possibly charged with degrading intercellular components. The stratum corneum is virtually devoid of phospholipids and is selectively enriched in ceramides (35%), free sterols and free fatty acids (25%), cholesterol sulfate (20%) with smaller quantities of glycolipids, sterol esters, triglycerides and hydrocarbons. Among all the epidermal lipids, glycosphingolipids are particularly suitable agents to help to fortify and replenish skin's moisture barriers and enhance hydration. These lipids participate in the regulation of cellular growth by maintaining proper intercellular communications. Also they have been proven to renew the skin's moisture binding capacity. Thus, depletion of lipids results in water loss and leads to skin damage.
The remainder of the minor epidermal cell populations is represented by melanocytes, Merkel cells, Langerhans cells, dendritic epidermal T cells, and epidermotropic lymphocytes. Melanocytes protect skin from UV radiation during the tanning process, Merkel cells do not have a defined function and other cells are distinct immunocompetent cells.
The second layer, dermis is a dense connective tissue layer that supports the first layer, epidermis. This layer consists of bundles of collagenous and elastic fibers. The former give firmness to the skin, the latter are rich in elastin and are responsible for the elasticity of the skin. Both, collagenous and elastic fibers form a reticular network to support the vasculature, nerves, lymphatics, and adnexal structures (hair, sweat glands, and such). Fibroblasts represent the major cell population within the dermis. Other cells within the dermis include; Mast cells, major inflammatory immunocompetent cell population, resident antigen-presenting cells, and transient inflammatory lymphoid cells such as polymorphonucleocytes, monocytes and lymphocytes.
Morphology of collagen and elastin plays an important role in the skin changes with aging process. (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 185-201) Elastin fibers largely consist of hydrophobic (non-polar amino acids) glycine, alanine, valine, proline sequences (elastic portions) alternating with hydrophilic (polar amino acid) lysine. Lysine residues always occur in pairs (Sandberg, L. B., Sockel, N. T.: N. Eng. J. Med. 1981, 304 (10), 566-579). These two cross-linking sites on different chains provide the possibility of the oxidative removal of the epsilon amino group followed by aldol condensation resulting into the pyridinium ring of desmosine formation. This crosslinking process results in the loss of elasticity (Sandberg, L. B., Sockel, N. T.: N. Eng. J. Med. 1981, 304 (10), 566-579). Also, during the aging process, a progressive fragmentation of elastic fibers occurs. As a result, the density of the fibrillar network in the dermis decreases. However, at the same time the surface density of skin elastic fibers increases with age due to the above described fragmentation and increased crosslinking activity (increased content of polar amino acids leads to higher number of crosslinking sites in elastin fibers) (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 185-201). Changes in the surface density together with fragmentation result in the loss of elasticity in the skin, and subsequently in appearance of fine lines and wrinkles, the visible signs of skin aging (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 185-201).
Destructive irreversible fragmentation of elastic fibers is well discussed in literature for pathological states of diseases such as rheumatoid arthritis, adult respiratory distress syndrome and pulmonary emphysema. The most widely accepted explanation of the observed pathogenesis is a proteinase-antiproteinase imbalance (Janoff, A., Carp, H.: Am Rev. Respir. Dis., 1985, 132, 417-434). This also applies for connective tissue proteins. Enzymes responsible for the destructive unwanted fragmentation of elastic fibers are proteases, namely elastases. Elastases are derived from many tissues in man including the pancreas, neutrophils, macrophages, monocytes, platelets, smooth muscle cells and fibroblasts. The ability to cleave elastin was found for thiol proteases such as papain, metalloproteases, such as macrophage elastase and serine proteases, such as leukocyte elastase (Reilly, C. F., Travis, J.: Biochem. Biophys. Acta, 1980, 621, 147-157).
Human leukocyte elastase (HLE) is a major serine protease that cleaves elastin and also cleaves type I, II, III, and IV collagens mimicking the action of collagenase. In particular, the powerful proteolytic activity of neutrophil elastase, HNE is essential for the migration of neutrophils through connective tissue for destruction of foreign bacterial invaders during the inflammation process. (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 23-31).
Under normal physiological conditions elastase levels are regulated by natural circulating plasma protease inhibitors. However, the elastase activity increases dramatically in an acute phase state and with age (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 23-31). An increase in HLE (HNE) activity within the dermis (fragmentation of elastic fibers followed by the loss of elasticity) plays a major role in skin aging, wrinkling. Similar properties have been detected for true collagenase, a metalloprotease that cleaves collagen. (Elastin and Elastases, Volume II, Eds. L. Robert, W. Hornebeck, CRC Press Inc., pages 57-62).
One of the primary "anti-aging" products on the present commercial market comprises elastin or collagen and thus provides a "substrate" for destructive proteases (elastase, collagenase). The addition of synthetic substrate to the skin treatment products will slow down the cleavage of the dermis' own elastin and collagen fibers. This type of skin treatment may increase the surface density of skin fibers in the upper dermis due to the accumulation of the fiber fragments and thereby, increases production of material that may become part of the stratum corneum, leading to excessive keratinization.
Other skin treatment product comprises pealing agents from the group of alpha-hydroxy acids, such as hyalluronic acid, glycolic acid etc. which peal and remove dead skin cells. Such formulations are useful to remove the excessive keratinization but they are not effective in terms of skin wrinkling treatment. However, they are used to clean the corneum stratum (the outer most layer of the epidermis) by loosening the bonds that hold the dead skin cells together, and thereby, giving a temporary smooth appearance to the skin.
As discussed above, water loss from epidermis is closely related to the depletion of lipids. Skin treatment formulations with Ceramide I (the lipidic ingredient closest to skin's own natural ceramides) helps to prevent moisture loss.
Another formulation for the treatment of skin wrinkles is based on a gel formulation comprising gelable hydrophilic polymer which fills up wrinkles and dries to impart to so-treated skin a smooth "wrinkle free" appearance.
While efforts have been directed in the past to design, develop and manufacture products that provide smooth appearance to the skin, none have been fundamentally targeting the destructive activity of proteases; a major factor contributing to skin changes with age. So far, no formulation on the market incorporates an agent which controls and/or reduces the increased protease (HLE and collagenase) activity. As shown above, proteolytic HLE and collagenase activity is responsible for the destruction of elastic and collagenous fibers and subsequently for skin wrinkling. If protease inhibitor, were provided in a skin delivery system, it would be possible to provide the desired proteinase-antiproteinase balance discussed above and slow down the process of skin aging.
The proteinase-antiproteinase balance requirement and replacement therapy is well described in literature for emphysema, arthritis, respiratory distress syndrome and other diseases. Replacement therapy, based on the introduction of natural or synthetic protease inhibitors received great attention in the scientific community. Researches have focused on the design and production of low and high molecular weight inhibitors (i.e. polymer bound inhibitor) of HLE and collagenase, some of which will be soon available on the market. To the best of the knowledge of the present inventors, however, no suggestion has been previously made in the art to use this technology to control skin wrinkles as now proposed.
The novel skin treatment delivery system and method of the present invention are directed to deliver protease (elastase, collagenase) inhibitor to the skin and create a thin long lasting occlusive film which controls the destructive activity of proteases and substantially stops the aging process, and that is both, effective and aesthetically pleasing. In addition, the composition of this invention maintains the sustained presence of inhibitor over a extensive period of time. Such topical compositions are also well suited for the treatment of inflammated areas.